Method of Providing Protective Immunity against Heterologous Leptospira Strains

ABSTRACT

The present invention provides compositions and methods for eliciting protective immunity in animals against  Leptospira Interrogans  (LI) serovar  copenhageni.  The invention is based, in part, on the unexpected cross-protection against heterologous LI serovar, which resulted when canines were administered an effective amount of RECOMBITEK® 4 Lepto, then subsequently challenged with virulent  L. copenhageni  (Fiocruz L1-130).

INCORPORATION BY REFERENCE

This application claims priority to provisional application U.S. Ser.No. 61/672,386, filed on Jul. 17, 2012, and incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to immunogenic leptospiracompositions, which are capable of eliciting cross-protective immuneresponses in animals, particularly canine animals. The invention furtherrelates to methods of providing animals, especially canine animals, withcross-protective immune responses against leptospira.

BACKGROUND OF THE INVENTION

Leptospirosis is an important world-wide zoonosis, caused by spirochetesfrom the Leptospira genus. It is an occupational hazard for many peoplewho work outdoors or with animals, including farmers, veterinarians,meat workers, dairy farmers, and military personnel. It is arecreational hazard for campers, or those who participate in outdoorsports in contaminated areas, and has been associated with swimming,wading, and whitewater rafting. Outbreaks of leptospirosis are usuallycaused by exposure to water contaminated with the urine of infectedanimals. Many different kinds of animals carry the bacterium; they maybecome sick but sometimes have no symptoms. Leptospira organisms havebeen found in cattle, pigs, horses, dogs, rodents, and wild animals,including marine mammals. Humans become infected through contact withwater, food, or soil containing urine from these infected animals. Thismay happen by swallowing contaminated food or water or through skincontact, especially with mucosal surfaces such as the eyes or nose, orwith broken skin.

The most common serovars reported in the United States are L.icterohaemorrhagiae, L. canicola, L. grippotyphosa, L. canicola and L.bratislava. Another serovar of interest reported in Latin America, is L.interrogans serovar Copenhageni. This serovar belongs to theIcterohaemorrhagiae serogroup, and has similarities in the DNA sequencefor known colonization virulence factors, and appears to be responsiblefor most canine leptospirosis in New Zealand.

REVIEW OF THE LITERATURE

“Leptospirosis Fact Sheet” (WHO, Regional Office for South-East Asia,2009) indicates, in part, that animals and humans can be immunized, butthat protection is largely serovar-specific. Lack of cross-protection isnot surprising, particularly in view of the significant genetic/genomicdifferences, for example, among the gene organization in thelipopolysaccharide biosynthetic (rfb) locus (Pena-Moctezuma, A. et al.,2001 FEMS Immunology and Medical Microbiology 31 (2001) 73-81).

Nascimento, A. L. T. O. et al. Comparative genomics of two Leptospirainterrogans serovars reveals novel insights into physiology andpathogenesis. Journal of Bacteriology, 186(7):2164-2172, 2004.

Nascimento, A. L. T. O. et al. Genome features of Leptospira interrogansserovar Copenhageni. Braz J Med Biol Res. 2004 Apr; 37(2).

Recombitek Lepto 4 (Merial Limited)—contains Leptospiraicterohaemorrhagiae (LI) was obtained from National Animal DiseaseCenter (NADC), Ames, Iowa, on 28 Feb. 1968 by Dow Chemical, Zionsville,Ind. The vaccine further contains Leptospira grippotyphosa, L. canicola,and L. pomona serovars.

Novibac (Merck Animal Health)—contains L. interrogans serogroup Canicolaserovar Portland-vere (strain Ca-12-000); L. interrogans serogroupIcterohaemorrhagiae serovar copenhageni (strain Ic-02-001); L.interrogans serogroup Australis serovar Bratislava (strain As-05-073);and L. kirschneri serogroup Grippotyphosa serovar Dadas (strainGr-01-005). Company-provided data indicates, unsurprisingly, theCopenhageni-containing vaccine elicits in canine an immune responseagainst serovar copenhageni. This product label claim is also consistentwith what is well-known in the Leptospira arts, namely, that little ifany evidence for “cross-protection”, which is herein defined asproviding protection against a heterologous Leptospira serovar byadministering an effective amount of a different serovar (e.g.protecting against copenhageni by administering a homologous effectiveamount of a non-copenhageni LI Icterohaemorrhagiae serogroup Leptospire.

Thus, as an alternative to vaccinating animals using all lepto serovarsagainst which immunological protection is desired, it would be useful toprovide new methods of eliciting cross-protective immune responsesagainst different lepto serovars. Until the instant disclosure, methodsfor providing protection against LI copenhageni using non-copenhageniserovars were not known.

SUMMARY OF THE INVENTION

An object of this invention is to provide methods for providingprotective immunity against a first Leptospira serovar comprising thestep of administering a second Leptospira serovar(s), which is from adifferent serogroup and/or serovar, with respect to the first Leptospiraserovar. In the cases where the second Leptospira serovar(s) is acombination of Leptospira serovars (e.g. a combination/multi-valentvaccine), the second Leptospira serovar(s) must not contain or comprisea Leptospira serovar of the same serovar as the first Leptospiraserovar, for which protective immunity is being sought.

In an embodiment, the methods provide protective immunity againstLeptospira Icterohaemorrhagiae (LI) serovar copenhageni, and comprisethe step of administering an effective amount of a non-copenhageni LIserovar to an animal in need thereof.

In a particular embodiment, the non-copenhageni LI serovar is from the24^(th) passage of LI obtained from National Animal Disease Center(NADC), Ames, Iowa, on 28 Feb. 1968 by Dow Chemical, Zionsville, Ind.

In another embodiment, the methods provide protective immunity againstLI serovar copenhageni by administering a combination/multivalentLeptospira vaccine. In a particular embodiment, the vaccine is Merial'sRECOMBITEK® 4 Lepto, as made in the United States as of Jun. 25, 2012.In an embodiment, the 4 Lepto comprises Leptospira canicola, Leptospiragrippotyphosa, Leptospira icterohaemorrhagiae, Leptospira pomona (allchemically inactivated) and has label claims (as of Jun. 25, 2012)according to the following: “provides protection against Leptospiragrippotyphosa for 15 months and prevents shedding of Leptospiraspirochetes in the urine. Specifically the vaccine is labeled to preventLeptospirosis and Leptospiruria caused by L. icterohaemorrhagiae, L.canicola, L. grippotyphosa. It also aids in the prevention ofLeptospirosis and Leptospiruria caused by L. Pomona.”

In a particular embodiment, and unexpected/surprising to the skilledworker in possession of the current state-of-the-art knowledge in thefield of leptospirosis, the invention provides for administration ofMerial's RECOMBITEK® 4 Lepto to canines to elicit protective immunityagainst a first LI serovar, which is not contained within the 4-wayvaccine, and which is LI serovar copenhageni.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof, to one of ordinary skill in the art, is set forthmore particularly in the remainder of the specification, includingreference to the accompanying figures, wherein:

FIG. 1 provides a graph of renal histopathology scores by group;

FIG. 2 is an image of kidney immunohistochemistry (40×) from a controldog with acute leptospirosis. Arrow indicates the presence of leptospiraspirochete in the renal tubules.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses methods for prevention of infectiondue to Leptospires of a particular serovar by administering Leptospiresof a different serovar.

In an embodiment, the invention provides methods of eliciting in ananimal a protective immune response against Leptospira interrogansserovar copenhageni comprising the step of administering to the animalan effective amount of a non-copenhageni Leptospira serovar.

In an embodiment, the non-copenhageni Leptospira serovar is delivered aspart of a multivalent/combination vaccine. In a particular embodiment,the non-copenhageni Lepto serovar is LI icterohaemorrhagiae.

In another embodiment, the vaccine comprises Leptospira Interrogans (LI)serovar icterohaemorrhagiae. In still another embodiment, the vaccinecomprises LI icterohaemorrhagiae, LI canicola, LI grippotyphosa, and LIpomona.

In a particular embodiment, the vaccine is Merial's RECOMBITEK® 4 Lepto,as manufactured in June 2012.

DESCRIPTIONS/DEFINITIONS

The “LI serovar Ictero” seed from RECOMBITEK® 4 Lepto is identifiedherein as ictero, which was obtained from National Animal Disease Center(NADC), Ames, Iowa, on 28 Feb. 1968 by Dow Chemical, Zionsville, Ind. On17 Dec. 1971, the seed was transferred to Pitman-Moore, Inc., LicenseNo. 264, Washington Crossing, N.J. A master seed was prepared by Dow atthe 20th passage. Pitman-Moore, Inc., produced a master seed after threepassages in artificial medium on 21 Jan. 1975 as “LI 1508 P23.” A masterseed was also qualified by Pitman-Moore, Inc., identified as “LI SC1518,P24 MS, Aug. 18, 1976.” Rhone Merieux, Inc. (now known as Merial, Inc.),License No. 298, received a culture from Pitman-Moore, Inc., on 30 Jun.1988 identified as “LI SC1518 P24 MS Aug. 18, 1976”.

The “LI serovar copenhageni” challenge strain was of different originand serogroup/serovar, when compared to the vaccination serovars(isolated from a human in Brazil, and designated: Fiocruz L1-130).

By “antigen” or “immunogen” means a substance that induces a specificimmune response in a host animal. The antigen may comprise a wholeorganism, killed, attenuated or live; a subunit or portion of anorganism; a recombinant vector containing an insert with immunogenicproperties; a piece or fragment of DNA capable of inducing an immuneresponse upon presentation to a host animal; a polypeptide, an epitope,a hapten, or any combination thereof. Alternately, the immunogen orantigen may comprise a toxin or antitoxin.

The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer can be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

The term “immunogenic or antigenic polypeptide” as used herein includespolypeptides that are immunologically active in the sense that onceadministered to the host, it is able to evoke an immune response of thehumoral and/or cellular type directed against the protein. Preferablythe protein fragment is such that it has substantially the sameimmunological activity as the total protein. Thus, a protein fragmentaccording to the invention comprises or consists essentially of orconsists of at least one epitope or antigenic determinant. An“immunogenic” protein or polypeptide, as used herein, includes thefull-length sequence of the protein, analogs thereof, or immunogenicfragments thereof. By “immunogenic fragment” is meant a fragment of aprotein which includes one or more epitopes and thus elicits theimmunological response described above. Such fragments can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996). For example, linear epitopes maybe determined by e.g., concurrently synthesizing large numbers ofpeptides on solid supports, the peptides corresponding to portions ofthe protein molecule, and reacting the peptides with antibodies whilethe peptides are still attached to the supports. Such techniques areknown in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysenet al., 1984; Geysen et al., 1986. Similarly, conformational epitopesare readily identified by determining spatial conformation of aminoacids such as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Methodsespecially applicable to the proteins of T. parva are fully described inPCT/US2004/022605 incorporated herein by reference in its entirety.

As discussed herein, the invention encompasses active fragments andvariants of the antigenic polypeptide. Thus, the term “immunogenic orantigenic polypeptide” further contemplates deletions, additions andsubstitutions to the sequence, so long as the polypeptide functions toproduce an immunological response as defined herein. The term“conservative variation” denotes the replacement of an amino acidresidue by another biologically similar residue, or the replacement of anucleotide in a nucleic acid sequence such that the encoded amino acidresidue does not change or is another biologically similar residue. Inthis regard, particularly preferred substitutions will generally beconservative in nature, i.e., those substitutions that take place withina family of amino acids. For example, amino acids are generally dividedinto four families: (1) acidic—aspartate and glutamate; (2)basic—lysine, arginine, histidine; (3) non-polar—alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar—glycine, asparagine, glutamine, cystine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another hydrophobicresidue, or the substitution of one polar residue for another polarresidue, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine, and the like; or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid that will not have a major effect on the biologicalactivity. Proteins having substantially the same amino acid sequence asthe reference molecule but possessing minor amino acid substitutionsthat do not substantially affect the immunogenicity of the protein are,therefore, within the definition of the reference polypeptide. All ofthe polypeptides produced by these modifications are included herein.The term “conservative variation” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

The term “epitope” refers to the site on an antigen or hapten to whichspecific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite”. Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to a composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, and/or cytotoxic T cells, directed specifically to an antigen orantigens included in the composition or vaccine of interest. Preferably,the host will display either a therapeutic or protective immunologicalresponse such that resistance to new infection will be enhanced and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms and/or clinicaldisease signs normally displayed by an infected host, a quicker recoverytime and/or a lowered viral titer in the infected host.

By “animal” is intended mammals, birds, and the like. Animal or host asused herein includes mammals and human. The animal may be selected fromthe group consisting of equine (e.g., horse), canine (e.g., dogs,wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domesticcats, wild cats, other big cats, and other felines including cheetahsand lynx), ovine (e.g., sheep), bovine (e.g., cattle), porcine (e.g.,pig), avian (e.g., chicken, duck, goose, turkey, quail, pheasant,parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g.,prosimian, tarsier, monkey, gibbon, ape), ferrets, seals, and fish. Theterm “animal” also includes an individual animal in all stages ofdevelopment, including newborn, embryonic and fetal stages.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a”, “an”, and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicate otherwise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

The invention will now be further described by way of the followingnon-limiting examples.

EXAMPLES Example 1 Cross-Protection of Recombitek® 4 Lepto against aLeptospira interrogans serovar Copenhageni Challenge in Dogs

Objective. To evaluate cross protection of RECOMBITEK® 4 Lepto against aLeptospira interrogans serovar Copenhageni challenge in dogs.

Materials and Methods. Twenty-four (24) purpose-bred beagles,approximately 2 months old, were randomly divided into two groups of 12dogs each. One group was administered Recombitek® 4 Lepto and the othergroup a placebo vaccine (PBS). All dogs received 2 subcutaneous doses (1ml) of the vaccine at a 21-day interval. Dogs from both groups werecommingled during the entire study. Approximately 4 weeks after thesecond vaccination all dogs were sedated and administered L. Copenhagenichallenge at 4.7 x 10⁸ lepto spirochetes/ml, 10 mls intraperitoneallyand 0.2 ml instilled topically per eye in the conjunctival sac.Following challenge, blood was collected periodically for liver profileand leptospira re-isolation. Sera samples were tested for serovarspecific antibody by microaglutination test at regular intervals. Urinewas collected periodically for re-isolation of leptospira. Dogs weresubject to necropsy at the end of the study and kidneys were harvestedfor histopathology and leptospira re-isolation.

TABLE 1 Study Design DOGS FRE- CHALLENGE* PER VACCINE ROUTE/ QUEN- (29Days GROUP GROUP GROUP DOSE CY post V2) A 12 Recombitek ® SC/1 ml Twice,Conjunctival 4 Lepto 21 days 0.2 ml/ apart Intraperitoneal B 12 Placebo(PBS) SC/1 ml Twice, 10 ml 21 days apart *Challenge dose: 4.7 × 10⁸organisms per ml, approximately 10^(9.73) organisms per dog.

Results. Dogs with mortality following L. Copenhageni challenge prior toplanned necropsies were classified as having acute leptospirosis if oneof the kidneys was positive for leptospirosis by immunohistochemistryand/or the histopathological lesions were indicative of acuteleptospirosis. Dogs that underwent necropsy at the end of the study wereclassified as having disease due to L. Copenhageni if it had one or morepositive urine samples and a renal histopathology score greater than orequal to 1 for either kidney.

TABLE 2 Incidence of disease, leptospiuria and leptospiremia Group Name(−) (+) Incidence of disease due to L. copenhageni challenge Placebo(PBS) 1 10 Test Vaccine (Recombitek ® 4 Lepto) 9  2 p-value of Fisher'sexact Test P ≦ 0.01 Leptospiuria - presence of leptospira in the urinePlacebo (PBS) 2  7 Test Vaccine (Recombitek ® 4 Lepto) 8  1 P-value ofFisher's exact Test P ≦ 0.05 Leptospiremia - presence of leptospira inthe blood Placebo (PBS) 1 10 Test Vaccine (Recombitek ® 4 Lepto) 9  1p-value of Fisher's exact Test P ≦ 0.01

TABLE 3 Clinical signs - incidence and duration post-challenge Totalnumber Mean duration Clinical Signs Group of dogs (days) DepressionPlacebo (PBS) 3/11 2.3 Recombitek ® 4 Lepto 0/11 NA Dehydration Placebo(PBS) 3/11 1   Recombitek ® 4 Lepto 1/11 1   Icterus Placebo (PBS) 3/113   Recombitek ® 4 Lepto 0/11 NA Conjunctivitis Placebo (PBS) 9/11 15.4 Recombitek ® 4 Lepto 5/11 7.8

Conclusions. This is the first time we report cross protection ofRecombitek® 4 Lepto against a L. Copenhageni challenge in dogs. Thesedata are both surprising and unexpected in view of the overwhelmingmajority of literature references, which collectively teach vaccinationwith a particular leptospira species does not provide protective immuneresponses against heterologous leptospira species (see especially the“Leptospirosis Fact Sheet” (WHO, 2009) and Pena-Moctezuma, A. et al.,2001). The incidence of leptospirosis, and leptospira re-isolation fromblood and urine was significantly higher in the placebo group incomparison to the Recombitek® Lepto 4 group. Dogs in the placebo grouphad higher incidence of depression, dehydration, icterus andconjunctivitis. All dogs in the placebo group had a renal histopathologyscore of 1 or greater. Mean ALP, ALT, BUN and creatinine values onspecific days were higher in the placebo group in comparison to theRecombitek® Lepto 4 group.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A method of providing an animal with protectiveimmunity against Leptospira interrogans serovar copenhageni comprisingadministering to an animal a vaccine comprising an effective amount of anon-copenhageni Leptospira serovar.
 2. The method of claim 1 wherein thevaccine is a multivalent/combination vaccine.
 3. The method of claim 2wherein the vaccine comprises Leptospira Interrogans (LI) serovaricterohaemorrhagiae.
 4. The method of claim 3 wherein the vaccinecomprises LI icterohaemorrhagiae, LI canicola, LI grippotyphosa, and LIpomona.
 5. The method of claim 3 wherein the vaccine comprisesRECOMBITEK® 4 Lepto, as manufactured in the United States in June of2012.
 6. The method of claim 3 wherein the animal is administered about1 ml of vaccine.
 7. The method of claim 3 wherein the animal isadministered 2 subcutaneous doses.
 8. The method of claim 7 wherein the2 doses are administered at a 21-day interval.
 9. The method of any oneof the proceeding claims wherein the animal is a canine.
 10. The methodof any one of claims 1 to 3 wherein the vaccine comprises additionalantigens that provide immunity against additional canine pathogens. 11.The method of claim 10 wherein the additional antigens are selected fromcanine parvovirus (CPV), canine parainfluenza virus (CPi2), caninedistemper virus (CDV), adenovirus, herpesvirus, rabies, caninecoronavirus, and combinations thereof.